We intend to produce somatic cell hybrids between 129 derived F9 mouse teratocarcinoma stem cells deficient in hypoxanthine phosphoribosyl-transferase and either human teratocarcinoma stem cells or mus caroli slpeen cells. The hybrids containing either the human or the mus caroli X chromosome will be studied for the expression of either human mus caroli X linked markers and for the expression of surface markers expressed by teratocarcinoma and early embryonal cells defined by monoclonal antibodies. Teratocarcinoma stem cell hybrids expressing the stage specific embryonal antigen 1 (SSEA-1) and lacking the expression of H-2 and Beta 2 microglobulin will be induced to differentiate with retinoic acid. We will then determine whether X chromosome inactivation occurs in vitro following cell differentiation and whether X chromosome inactivation is random or whether it affects preferentially either the 129 or the mus caroli or the human X chromosome. By taking advantage of the availability of X choromosome specific nucleic acid probes for the genes for glucose-6-phosphate (G6PD) and hypoxanthine phosphoribosyltransferase (HPRT) we will determine: 1) whether there is an inverse correlation between methylation of the G6PD and HPRT genes and G6PD and HPRT activity in stem and differentiated cells, 2) whether the transcription of the inactive X chromosome is shut off in differentiated cells; and 3) whether methylation preceeds or follows the suppression of the transcription of X linked markers. This study shoud result in a better understanding of the molecular mechanisms resulting in X chromosme inactivation in mammalian cells.